1. Field of the Invention
The present invention relates generally to the delivery of gene medicines to the brain and the eye. More particularly the present invention involves the combination of liposome technology, blood-brain barrier (BBB)/blood-retinal barrier (BRB) receptor technology, pegylation technology, and therapeutic gene technology to provide formulations which are useful in the non-invasive delivery of genes to the brain and the eye.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
The expression of exogenously administering genes in brain has previously been achieved in vivo with either viral vectors or cationic liposomes (1–4). However, in either case, highly invasive routes of administration are required. Such invasive techniques are needed because of the failure of either viruses or cationic liposomes to cross the brain capillary wall, which forms the blood-brain barrier (BBB) in vivo. The existence of the BBB necessitates the administration of the exogenous gene either intracerebrally via craniotomy (1), or by the intra-carotid arterial infusion of noxious agents that cause BBB disruption and transient opening of the BBB (4).
Human gene therapy of the brain will likely require repeated administration of the gene medicine. Therefore, it would be advantageous to administer the gene by a route that is no more invasive than a simple intravenous injection. With this approach, the gene therapeutic is delivered through the BBB by targeting the gene medicine to the brain via endogenous BBB transport systems (5). Carrier-mediated transport (CMT) systems exist for the transport of nutrients across the BBB (5). Similarly, receptor-mediated transcytosis (RMT) systems operate to transport circulating peptides across the BBB, such as insulin, transferrin, or insulin-like growth factors (5). These endogenous peptides can act as “transporting peptides,” or “molecular Trojan horses,” to ferry drugs across the BBB. In this approach, called the chimeric peptide technology, the drug that is normally not transported across the BBB is conjugated to a “transportable peptide, and the drug/transportable peptide conjugate undergoes RMT through the BBB (U.S. Pat. No. 4,801,575).
Peptidomimetic monoclonal antibodies (MAb) that bind endogenous transport systems within the BBB, such as the transferrin receptor (TfR) or insulin receptor, have been used in previous studies for targeting neuropeptides or antisense agents through the BBB in vivo (5). The ability of certain receptor-binding MAbs to mimic the action of the endogenous peptide that binds the same receptor is well known in the literature (31–33). In addition, the ability of such peptidomimetic MAbs, such as anti-TfR MAbs, to transport drugs into cells via these receptor-mediated endocytosis is also well known (34).
The expression in the brain of a therapeutic gene requires that the gene formulation that is injected into the blood is transported not only across the BBB by RMT, but also across the brain cell membrane (BCM) by receptor-mediated endocytosis (RME) into the target cell in brain. In addition, using endogenous BBB transport systems to target gene medicines non-invasively to the brain also requires the development of a suitable formulation of the gene therapeutic that is stable in the bloodstream. Cationic liposome/DNA complexes have been used for in vivo gene expression, but these formulations aggregate extensively in saline solution (6–11). This aggregation results in selective gene expression in the lung with little expression in peripheral tissues (12–14), and no expression in brain following intravenous administration of the cationic liposome/DNA complex (12). The DNA plasmid could be conjugated to the peptidomimetic MAb via a cationic polylysine bridge (15–17). However, electrostatic interactions between DNA and polycations may not be stable in blood, and highly polycationic proteins such as histone or polylysine exert toxic effects at the BBB and cause generalized BBB permeability changes in vivo (18).
Human blindness is a surprisingly common disorder that afflicts a large number of individuals (46). Approximately 10% of the population over 60 years of age suffers from macular degeneration, and approximately 1 in 3,000 births results in a mutation in a gene that plays an important role in vision (35). These inherited forms of blindness are collectively referred to as retinitis pigmentosa (RP). There are over 100,000 individuals in the U.S. alone that suffer from RP. The various RP genetic disorders can be traced to mutations in over 100 different genes with mutations in the rhodopsin gene comprising about 10% of RP (36).
In most cases, the mutated gene that causes RP is known, and the gene has been cloned. Therefore, gene discovery is no longer the rate-limiting issue in the gene therapy of RP. Rather, the rate-limiting problem is how to target the therapeutic gene throughout the entire retina. Present day forms of gene therapy to the retina involves packaging a therapeutic gene in a viral vector, such as adenovirus, herpes simplex virus (HSV), or adeno-associated virus (AAV). Next, the virus carrying the therapeutic gene is injected directly into each eye as either an intra-vitreal injection, or more commonly as a sub-retinal injection. The area of the retina that is tranduced with the virus is only at the tip of the injection needle. Areas of retina that are only 100 μm, which is thickness of human hair, away from the injection needle are not transduced with the exogenous gene (35).
There are at least two problems with the existing forms of gene therapy of retinal diseases. First, viral vectors are used. Virtually all humans have a preexisting immunity to either adenovirus or HSV, and the injection of these viruses into the central nervous system causes severe inflammation, which essentially aborts the therapeutic effect of the gene (36–45 and 25). More recently, AAV has been used as the viral vector because humans are less likely to have a preexisting immunity to this virus. However, single injection of AAV into the eye of a primate results in the development of high-tier neutralizing anti-AAV antibodies in the bloodstream (35). The development of these anti-viral neutralizing antibodies sets the stage for inhibiting the therapeutic effect of the subsequent gene treatments, and may also cause local inflammation within the eye.
The second problem with the existing forms of retinal gene therapy relates to the fact that the virus does not cross the blood-retinal barrier (BRB). Therefore, the virus cannot access the retina following non-invasive (intravenous, subcutaneous) forms of administration. Consequently, the viral vector carrying the exogenous gene must be administered to the retina or other structures of the eye by direct injection into the eye. This only allows for viral transduction within cells at the tip of the injection needle, which in turn would create a “pin-hole” visual field for the patient. Therefore, to have a wider distribution of the exogenous gene in the retina, it would be necessary to make multiple injections in both eyes. This would be an invasive procedure that may not be widely adapted to hundreds of thousands of patients with RP, and would also create an aberrant ‘multiple pin-hole’ field of vision for the patient. As is apparent, there is a present need to provide a non-viral non-invasive targeting technology for delivering therapeutic genes and other pharmaceutical agents to the entire retina.